2021年10月17日 星期日
黄毛草莓编码NB-ARC结构域的FnCN基因和启动子克隆及FnCN基因表达分析
Cloning of FnCN gene encoding NBARC domain and its promoter from Fragaria nilgerrensis and analysis on FnCN gene expression
2020年 第29卷 第4期 页码[1-10]    下载全文[6.4MB]  
摘要

利用同源克隆技术得到黄毛草莓(Fragaria nilgerrensis Schlecht. ex Gay)中编码NB-ARC结构域的抗病相关基因FnCN及启动子序列。序列分析结果表明:FnCN基因开放阅读框的长度为933 bp(GenBank登录号MN240290),编码310个氨基酸残基,其N端有1个Rx-CC结构域,C端有1个保守的NB-ARC结构域。该基因起始密码子上游启动子pFnCN序列的长度为910 bp(GenBank登录号MN240291),包含TATA-box、CAAT-box、激素响应元件、干旱诱导MYB结合位点、光响应元件和厌氧诱导顺式作用元件等。氨基酸序列的同源性比对结果表明:黄毛草莓FnCN基因与野草莓(Fragaria vesca Linn.)CN基因编码氨基酸序列的相似性最高(95.16%)。成功构建了黄毛草莓FnCN基因的植物过表达载体pCAMBIA1301-HA-FnCN和病毒诱导基因沉默载体pTRV2-FnCN。实时荧光定量PCR检测结果表明:黄毛草莓叶片接种胶孢炭疽菌〔Colletotrichum gloeosporioides (Penz.) Sacc.〕后0~3 h FnCN基因表达下调,之后逐渐上调;接种后48 h该基因的相对表达量最高,是接种后0 h的3.3倍。研究结果显示:黄毛草莓FnCN基因响应胶孢炭疽菌的胁迫,推测其在黄毛草莓抗炭疽病过程中发挥着重要作用。

Abstract

Sequences of disease-resistant related gene FnCN encoding NB-ARC domain and its promotor were obtained from Fragaria nilgerrensis Schlecht. ex Gay by using homologous cloning technology. The sequence analysis result shows that the length of open reading frame. of FnCN gene is 933 bp (GenBank accession number MN240290), which encodes 310 amino acid residues, including a Rx-CC domain at Nterminal and a conserved NB-ARC domain at C-terminal. The sequence length of up-stream promotor pFnCN of start codon of FnCN gene is 910 bp (GenBank accession number MN240291), which includes cis-acting elements such as TATA-box, CAAT-box, hormone responsive element, MYB binding site in drought induction, light responsive element, and cis-acting element in anaerobic induction, etc. The homologous alignment result of amino acid sequence shows that amino acid sequence encoded by FnCN gene from F. nilgerrensis has the highest similarity (95.16%) with that encoded by CN gene from Fragaria vesca Linn. The plant overexpression vector pCAMBIA1301-HA-FnCN and virus induced gene silencing vector pTRV2-FnCN of FnCN gene from F. nilgerrensis are successfully constructed. The detection result of real-time fluorescence quantitative PCR shows that FnCN gene expression in leaf of F. nilgerrensis is down-regulated at 0-3 h after inoculation of Colletotrichum gloeosporioides (Penz.) Sacc., and then gradually up-regulated. The relative expression of FnCN gene is the highest at 48 h after inoculation, which is 3.3 times that at 0 h after inoculation. It is suggested that FnCN gene of F. nilgerrensis can response to C. gloeosporioides stress, indicating that it may play an important role in anti-anthracnose process of F. nilgerrensis.

关键词黄毛草莓; 炭疽病; NB-ARC结构域; FnCN基因; 实时荧光定量PCR
Key wordsFragaria nilgerrensis Schlecht. ex Gay; anthracnose; NB-ARC domain; FnCN gene; real-time fluorescence quantitative PCR
作者胡玉慈1, 姚立萍2, 张童1, 何淑敏3, 唐鑫彪1, 陈清西1, 文志丰1
所在单位1. 福建农林大学园艺学院, 福建 福州 350002; 2. 福州市农业科学研究所, 福建 福州 350018;3. 中国农业科学院蔬菜花卉研究所, 北京 100081
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基金项目国家自然科学基金(青年项目)(31701907); 福建省自然科学基金项目(2018J01703); 福建农林大学杰出青年科研人才计划项目(xjq201723); 福建省教育厅中青年教师教育科研项目(JAT170163); 福建省现代农业产业技术体系建设专项(闽财指[2017]625号)